Chagas disease is a leading cause of morbidity and mortality in endemic areas, mostly because approximately 30% of patients progress to inevitably fatal chronic cardiomyopathy. It is caused by the obligate intracellular parasite Trypanosoma cruzi, which inhabits the cytosol of parenchymal cells in the heart and other organs, where it expands and maintains infection through intracellular differentiation and multiplication cycles. To reach the host-cells, T. cruzi must migrate through the endothelium otherwise it will be trapped, and likely die, in the bloodstream. Yet mechanisms governing T. cruzi transendothelial migration (TEM) remain unknown. The goal of this R21 application is to gain insights into T. cruzi TEM by testing the hypothesis that T. cruzi takes advantage of endothelial cell programming used by leukocytes for extravasation in inflamed tissues. The hypothesis is strongly supported by preliminary results related to T. cruzi-heart endothelial cell interaction. The proposed project is an extension of those reproducible pilot experiments. Specifically, the goal is (Aim 1) to assess T. cruzi induced upregulation of endothelial apical adhesion receptors physiologically intended for leukocyte usage, and if those adhesion receptors promote T. cruzi attachment to the endothelium; and (Aim 2) to ascertain biological relevance of T. cruzi binding to endothelial apical adhesion receptors by evaluating T. cruzi paracellular and transcellular endothelial migration using fluorometric and state-of-the-art visualization procedures. Aside from mechanisms, this project may unveil unique therapeutic opportunities for Chagas disease, as blockage of T. cruzi extravasation will likely reduce tissue parasitism, like drug development efforts to prevent leukocyte extravasation in autoimmune diseases. One of the project co-PIs is expert in mechanism of T. cruzi-host cell recognition, and the other has extensive experience in leukocyte extravasation mechanisms in inflammation and autoimmune diseases. Their expertise will be combined synergistically to accomplish each of the two Aims and ensure the project's success.